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Accepted Preprint first posted on 7 April 2014 as Manuscript JME-13-0238

For submission to “Journal of Molecular Endocrinology: Revision 1”

Origin and Functional Evolution of the Corticotrophin-Releasing Hormone Receptors

David A. Lovejoy1, Belinda Chang1,2, Nathan Lovejoy 3, Jon del Castillo1 1.Department of Cell and Systems Biology, University of Toronto, 2. Department of Ecology and Evolution, University of Toronto, 3. Department of Life Sciences, University of Toronto, Scarborough

Corresponding Author:

Dr. David A. Lovejoy Department of Cell and Systems Biology 25 Harbord Street University of Toronto Toronto, Ontario L4A IK6 Canada Telephone: 416 946 7259 Email: [email protected]

Number of Pages: 35 Number of Figures: 6 Keywords: stress, Metazoa, diuresis, energy metabolism, reproduction, central nervous system

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Copyright © 2014 by the Society for Endocrinology.

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Abstract Corticotrophin-releasing hormone (CRH) is the pivotal neuroendocrine peptide hormone associated with the regulation of the stress-response in vertebrates. However, CRH-like peptides are also found in a number of invertebrate species. The origin of this peptide can be traced to a common ancestor of lineages leading to chordates and to arthropods, postulated to occur some 500 million years ago. Evidence indicates the presence of a single CRH-like receptor, and soluble binding protein system that acted to transduce and regulate the actions of the early CRH peptide. In vertebrates, genome duplications led to the divergence of CRH receptors into CRH1 and CRH2 forms in tandem with the development of four paralogous ligand lineages that included CRH; urotenin-1/urocortin, urocortin 2 and urocortin 3. In addition, taxon-specific genome duplications led to further local divergences in CRH ligands and receptors. Functionally, the CRH ligand-receptor system evolved initially as a molecular system to integrate early diuresis and nutrient acquisition. As multicellular organisms evolved into more complex forms, this ligand-receptor system became integrated with the organismal stress response to coordinate homeostatic challenges with internal energy usage. In vertebrates, CRH and the CRH1 receptor became associated with the hypothalamo-pituitary-adrenal/interrenal axis and the initial stress response, whereas the CRH2 receptor was selected to play a greater role with diuresis, nutrient acquisition and with the latter aspects of the stress-response.

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1. Introduction

The evolution of the corticotrophin-releasing hormone (CRH) family of peptides and their cognate receptors provides a model to understand peptide ligand and receptor co-evolution. Over the last few years with the acquisition of genomic data from numerous metazoan species, our understanding of the CRH ligand-receptor system has become much clearer. It has subsequently allowed us to explain why ligand gene duplications do not necessarily corroborate with receptor gene duplications. In addition, such studies help establish how physiological functions, associated with gene expansion events in vertebrates relates to the ancestral invertebrate genomes. With respect to CRH physiology, these genomic studies shed light on why peptides, initially associated with diuresis and feeding become associated with the stress response as we know it. Thus, the goal of this review is to understand the evolutionary history and functional expansion of the CRH receptors, and why two such receptor paralogues have become selected for the regulation of the stress response in vertebrates, where four ligand paralogues exist. Elucidation of the CRH family and its receptors has its origins with the earliest forays into our understanding of neuroendocrinology in the first part of the 20th century. Its history is intrinsically tied to the understanding of receptor-ligand interaction and gene duplication paradigms (Lovejoy and Balment, 1999; Lovejoy 2005; Lovejoy and Jahan, 2006, Lovejoy 2009). However, establishing a universal approach to receptor-ligand structure and function in the current scientific environment requires some reconciliation with two complementary, yet frequently misunderstood philosophical approaches. On one hand, a comparative and evolutionary approach, where structure-function studies performed using numerous species across the Metazoa provides an understanding of the origin, conservation and evolution of the receptor-ligand system in question. On the other hand, should a particular ligandreceptor system have applications to human health then, a research approach that utilizes fewer species that act as models to understand human health and pathology is typically initiated. The scientific goals of both approaches are essentially similar, however, the terminology used may generate confusion among researchers associated with either approach. In this review, we have focused on the first approach utilizing biomedical research insofar that it helps establish the reasons for the origin, evolution and function of the CRH receptors. The evolution and function of vertebrate neuroendocrine pathways is complex. The effect of speciation throughout numerous niches and habitats, coupled with widespread gene and genome 3

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expansion events has led to the formation of functionally-related paralogous ligands and receptors, as well as divergent orthologues. Thus, the delineation of a single neuroendocrine pathway can be a daunting task. The classical interpretation is that the ligand and its receptor arguably play the greatest role in determining the specificity of action. However, because of the relative structural simplicity of a ligand in comparison to its receptor, historically, the discovery and structural characterization of the ligand has preceded the elucidation of the receptor mechanism. As with most neuroendocrine systems, the discovery, structural characterization and functional attributes of the CRH receptors occurred later than the onset of the CRH ligand discoveries. Early attempts at the purification of receptors provided limited information on their structures. G-protein-coupled receptors (GPCR) are particularly difficult to solubilise and isolate because of their complex association with the plasma membrane. Thus, it was not until the advent of mRNA isolation and cloning methods could these receptors be characterized. Although the first CRH-related ligand was discovered in 1979 (Montecucchi et al., 1979), it was not until Chen and her associates in Wylie Vale’s laboratory elucidated the structure of the first CRH receptor (Chen et al., 1993). Since then, the identification of numerous CRH paralogues and orthologues, combined with the pharmacological and physiological evaluation of their actions with the additional modern genome sequencing methods, has led to a detailed model of CRH receptor structural and functional evolution.

2. Discovery, Evolution and Function of CRH Peptides The first CRH, per se, purified from sheep hypothalami was published by Wylie Vale and associates in 1981, well after the initial evidence that provided its existence in 1955 was reported (Schally and Saffran, 1955; Guillemin and Rosenberg, 1955). However, a few years before the report of the CRH structure, Montecucchi and coworkers (1979) identified the structure of sauvagine (SVG), a 41KDa peptide from the skin of neotropical frog (Phyllomedusa sauvagei). Later, a similar peptide was characterized in the urophysis of a fish species (white sucker, Catostomus commersoni) and was called urotensin-I (UI; Lederis et al., 1982), to distinguish it from urotensin-II also found in the urophysis but structurally unrelated to the CRH family of peptides. Both UI and SVG were shown to be effective at stimulating ACTH release from the rat pituitary gland although their pharmacological profiles differed from that of CRH (see Lovejoy and Balment, 1999; Lovejoy 2009 for reviews). As a result, these peptides became important tools in the identification the pharmacological properties of the CRH receptors once they were discovered. The mammalian orthologue of UI and SVG was reported in 1995 (Vaughan et al., 1995) and was named urocortin (Ucn), thus confirming the conservation of two paralogous CRH-like 4

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peptides in the vertebrates (see Lovejoy and Balment, 1999). However, it is important to note, that from an evolutionary perspective, UI and Ucn are paralogues of CRH and physiologically, they should be treated as such, rather than grouping them with the later discovered Ucns 2 and 3 (Fig.1). In 1989, a peptide hormone associated with diuresis in the tobacco hornworm (Manduca sexta; Kataoka et al., 1989) was characterized and showed remarkable structural similarity to the CRH peptides, notably SVG. Its high degree of residue identity indicated that it was homologous to the vertebrate CRHs. This discovery was followed by additional reports of orthologous CRH-like peptides, collectively referred to as the diuretic hormones, in insects (Coast, 1998). However, these peptides shared significant structural similarity with all of the vertebrate CRH paralogues with no clear evidence of separate CRH, and UI/Ucn forms (Lovejoy and Jahan, 2006). This mystery was resolved with the characterization of a peptide with structural similarity to the vertebrate CRH and urocortins, on one hand and the insect diuretic hormones on the other in the tunicates, Ciona intestinalis and C. savignii (Lovejoy and Barsyte Lovejoy, 2010). Because the tunicates (urochordates) are the evolutionarily closest sister lineage to the Chordata (Delsac et al., 2006), this report supported the concept that only a single CRH peptide was inherited by the ancestral vertebrates. Further studies (see below) indicate that this peptide was not initially involved in a vertebrate-like ‘hypothalamus-pituitary-adrenal/interrenal (HPA/I)”-like axis. In vertebrates, as postulated by the 2R hypothesis, which stated that there were two rounds of genome duplication at the base of vertebrate evolution four CRH paralogues should exist (Dehal and Boore, 2005), if there was a single CRH-like gene inherited. The independent work of the Vale (Lewis et al., 2001, Reyes et al., 2001) and Hseuh laboratories (Hsu and Hseuh, 2001) confirmed this with the characterization of Ucn 2 (stresscopin) and Ucn 3 (stresscopin-like peptide) (Fig 1).

Further

characterization of CRH paralogues in vertebrates have confirmed the presence of four CRH-like peptide genes among jawed vertebrates, although Ucn 2 appears to be non-functional in the elephant shark (Callorhynchus milii; Nock et al., 2011) and chimpanzee (Ikemoto and Park, 2006) because of the lack of conserved initiation codons suggesting that the prohormones are not produced. Indeed, the strong structural similarity between Ucn 2 and 3, has led to the interpretation that one or more of the functions of Ucn 2 has been taken over by Ucn 3 in these species. Nevertheless, studies on the expression of CRH peptides in the Metazoa indicated that a single form was inherited by the basal vertebrates when the first genome duplication led to the formation of a CRH/urotensin-I/urocortin ancestral peptide on one hand, and a urocortin 2/3 ancestral peptide on the other hand. The second 5

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duplication event led to the formation of the four individual peptides (Lovejoy and Jahan, 2006; Lovejoy, 2009; de Lannoy and Lovejoy, 2013) (Figs 2,3). Further, all four peptide genes are found on different chromosomes (see Lovejoy 2012), consistent with the original hypothesis of the relationship between chromosomal and paralogon duplication and the 2R hypothesis (Delsac et al., 2006; Lundin, 1993; Holland et al., 1994; Holland 1999). 3.

Description of CRH family of peptides and their functions.

Studies conducted primarily in insect models, where only a single CRH-like peptide (diuretic hormones; DH) is present, suggest that the earliest function of the peptide family was associated with diuresis and feeding (Kataoka et al., 1989; Audsley et al., 1997; Coast, 1997; 1998). Given the phylogenetic age of the CRH peptides, this is not surprising. In early metazoans, because of their less complex physiology and genome, extracellular regulatory systems were ultimately tied to the coordination of energy production to survival with respect to feeding, digestion, diuresis and defence primarily, and secondarily, reproduction. Thus, the formation of the CRH-like peptides in ancestral metazoans may have been selected and conserved through evolution because they acted to regulate the utilization of cellular and organismal energy acquisition and production for defence against environmental stressors. This has come to be known as the organismal stress-response that acts to protect the organism from external and internal environmental challenges.

4. Discovery, Structure and Evolution of CRH Receptors

Although the original discovery of an active corticotrophin-releasing factor necessitated that at least one type of receptor was present, it was not until the first comparative pharmacological studies of ovine CRH, frog SVG and fish UI were performed that evidence of additional classes of binding sites became known. In 1985, Lederis and associates suggested that, based on the comparative affinities of CRH, UI and SVG in a number of different tissue types, the mammalian vascular receptors were different from those on the mammalian pituitary corticotropes and further postulated that the mammalian vascular and fish pituitary receptors may be similar, whereas the mammalian pituitary receptors are different. Attempts to characterize the CRH receptor began shortly after the discovery of ovine CRH (Vale et al., 1981). High-affinity CRH-binding sites were subsequently established in rat and human pituitary glands and brains (Wynn et al., 1983; DeSouza et al., 1985; DeSouza et al., 1984; DeSouza et al. 1986), as 6

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well as in the corticotrope cell line AtT20 (Rosendale et al., 1987). Moreover, this receptor was shown to be associated with a cAMP-dependent signal transduction system (Bilezikjian and Vale, 1983; Labrie et al. 1982; Aguilera et al., 1983) likely via a G-protein (Perrin et al., 1986) (Fig. 2). Attempts to purify the receptor showed a molecular weight of 40-45 kDa (Nishimura et al., 1987; Grigoriadis and DeSouza, 1989). However, numerous attempts to solubilise and purify the receptor for amino acid sequencing analyses were ultimately unsuccessful. A soluble high affinity 37-kDa CRH binding protein was, however, isolated and partially characterized from human plasma (Behan et al., 1989) and cloned shortly after (Potter et al., 1991). However, the structure of this protein was unique and did not possess any attributes suggesting that it may be related to the sought after membrane bound receptor. The first CRH receptor (CRH1) was reported by Chen and colleagues (1993) after preparing an expression library from a human corticotropic tumour. This landmark publication defined the archetype of the CRH receptor. The cloned cDNAs encoded a receptor of 415 residues and a second splice variant with a 29-residue extension. Further cloning studies indicated that the CRH1 receptor showed considerable structural variability and varied from 415 to over 440 residues (Hauger et al., 2006; Pohl et al., 2001; Arai et al., 2001; Huissing et al., 2004). For example, the human CRH1 gene possesses 14 exons spanning 20 kb (Sakai et al., 1998), whereas the rat gene possesses only 13 exons (Tsai-Morris et al., 1996). In the Japanese pufferfish (Fugu rubripes) this receptor consists of 14 exons encompassing 27 kb over the genomic sequence (Cardoso et al., 2003). The CRH1 receptor was established to be a member of the guanine protein (G-protein) coupled receptors (GPCR) family (Chen et al., 1993), one of the largest and most studied families of receptors. Their signature characteristic is the presence of a highly conserved seven-transmembrane domain (Cardoso et al., 2006). They bind to a variety of ligands including protons, odorants, biogenic amines, peptides and glycoproteins resulting in a variety of G-protein mediated effects (Ulloa-Aguirre et al., 1999; Fredriksson et al., 2003). The CRH1 and subsequent paralogues (see below) were established to belong to the Family B GPCRs and share the common characteristic of a long N-terminal domain and a highly glycosylated external domain (Attwood and Findlay, 1994; Fredriksson et al., 2003; Harmar, 2001). Ligands for B Family receptors also include vasoactive intestinal peptide (VIP), calcitonin, parathyroid hormone, secretin, growth hormone releasing hormone (GRH), glucagon and pituitary adenylate cyclase activating peptide (PACAP) (Fredriksson et al., 2003). This family of receptors is particular important for the regulation of ion and nutrient transport as well as playing a role with various elements of the stress response (Fredriksson et al., 2003, Harmer, 2001; Harmar et al., 2012).

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The origins of this group of receptors can be traced to a period before the bifurcation of deuterostomes and protostomes, over 500 million years ago. However, because this GPCR receptor family is particularly well established in chordates (deuterostomes) (Fredriksson et al., 2003) and arthropods (protostomes) (Kwon et al., 2012; Li et al., 2013; Veenstra et al., 2012), its earliest origins may be much earlier. The first prototypes of this receptor family may have begun during the beginning of bilateral animal evolution as there is no clear evidence of this receptor family in basal metazoans such as Placozoa, Porifera, Ctenophora or Cnidaria. The early evolution of the Family B GPCR family and, therefore, the earliest CRH receptors agrees with the postulated time of origin for CRH-like peptides (Lovejoy and De Lannoy, 2013; Lovejoy and Balment, 1999; Lovejoy and Jahan, 2006). Of the Family B receptors, the CRH receptors show the closest structural similarity with the calcitonin (amylin, guanylin, calcitonin-gene related peptide) family of receptors (Fredriksson et al., 2003). This relationship among receptors is also reflected by the high sequence similarity of the insect calcitonin-related peptide, DH31, with Ucns 2 and 3, and also the tunicate CRF-like peptide (Coast et al., 2001; Kwon et al., 2012; Lovejoy and Barsyte-Lovejoy 2010; Lovejoy and Jahan 2006). Moreover, the DH31 peptide binds and activates a calcitonin-like receptor in insects where it plays a diuresis role (Brugge et al., 2008; Christie et al., 2010; Furuya et al., 2000; Kwon et al., 2012; Zandawala, 2012; Zandawala et al., 2011). The CRH1a receptor is the dominant subtype that is widely expressed in the mammalian brain and a number of peripheral tissues (Hillhouse and Grammatopoulos, 2006) and is associated directly with the organismal coordination of the HPA/I associated stress response. Given the evolutionary selection and conservation of this mechanism, it is perhaps not surprisingly that a number of alternatively spliced tissue-specific forms that show modified signal transduction ability have also been identified that act to modify the elements of the stress-response (Chen et al., 2003; Hauger et al., 2006). An integrated understanding of the combined role of these receptor subtypes is not entirely understood, but may act in part to provide a “fine-tuning” of the CRH1 mediated stress response. For example, the CRH1d variant, which shows impaired cellular signalling ability, may act as a decoy receptor that competes with CRH1a to modify the response due to incoming CRH or Ucn-mediated signals (Hillhouse and Grammatopoulos, 2006) although this has yet to be conclusively established. The CRH2 receptor was reported shortly after the discovery of the CRH1 receptor. The identification and structural characterization of the CRH2 receptor was reported in mouse (Kishimoto et al, 1995; Perrin et al., 1995; Stenzel et al., 1995),rat (Lovenberg et al., 1995) and human (Liaw et al., 1996). The designation of CRH1 and 2 receptors by Lovenberg and colleagues in 1995, was ultimately adopted as the international nomenclature of the CRH receptors. There are three recognized splice 8

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variants (CRH2a, b, c) and two truncation variants of the CRH2 receptor found in mammals, but only the CRH2a variant has been clearly identified in non-mammals (Arai et al., 2001; Hauger et al., 2006, Hillhouse and Grammatopoulos 2006). The receptors can be distinguished on the basis of the amino acid sequence of their amino terminal domain. In addition, the CRH2a receptor form was initially reported to be processed as a soluble binding-protein that consists of the 1st extracellular domain (Chen et al., 2005) however, later studies indicated that this truncated mRNA could not be secreted due to an ineffective signal peptide (Evans and Seasholtz, 2009). The CRH2 gene consists of 15 exons encompassing about 50 kb in the genome. The first four exons encode the different 5’ ends of the receptor and the remaining exons encode the remaining parts of the receptor (Catalano et al., 2003; Hauger et al., 2006; Pohl et al., 2001). Generally, orthologues among both CRH1 and CRH2 receptors are about 80% or more identical with each other at the amino acid level, whereas there is about 70% residue sequence identity among paralogues. However, there is only about 30% identity of the CRH receptors with other members of the Family B GPCRs. The greatest level of sequence identity occurs among the intracellular and transmembrane regions, whereas the extracellular regions are more variable. The 3rd intracellular loop, which possesses the G protein interacting site is the most highly conserved, with the identity approaching 100% in mammalian orthologues (Hauger et al., 2006). For example, the rhesus monkey (Macaca mulatta) possesses 99.5% identity at the amino acid level with humans, and as expected, has a similar pharmacological profile with the human CRH1 receptor (Oshida et al., 2004). Other types of CRH-binding receptors and proteins have been identified. A third CRH receptor (CRH3) was found in the catfish, Ameriurus nebulosa (Arai et al., 2001). However, because of the close structural and pharmacological similarity between the catfish CRH1 and CRH3 receptors, it was likely that the third receptor is the result of the extra genome duplication in occurs in a number of teleost lineages (Mulley et al., 2009; Mugpakdee et al., 2008; Wolfe et al., 2001) including for example, the white sucker (Morley et al., 1991), goldfish and rainbow trout (Doyon et al., 2003) where two CRH peptides are present. Interestingly, two CRH forms have been identified in the chondrichthyan, C. milii, although the identification of the CRH receptors has not been established (Nock et al., 2011). Regardless, the selection pressure in other taxonomic groups of fishes led to the retention of this extra receptor, although the reasons for this are not entirely understood. The CRH-binding protein, which is structurally distinct from the CRH receptors, is a 37 kDa N-linked glycoprotein which binds to both CRH and Ucn and its orthologues (Sutton et al., 2005; Valvede et al., 2001) although no clear paralogues of this protein have been identified (Behan et al., 1989; Potter et al. 9

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1991, Huising et al 2004, 2005). The role of the binding protein is unclear. Comparative binding studies indicates that it possessed a greater affinity for UI,SVG (Sutton et al., 1995) and later Ucn (Vaughan et al., 1995). Orthologues are of this protein are well established with the insect lineages. Such conservation indicates that the binding protein evolved early in the evolution of CRH/DH ligand receptor systems and has become an integral part of the CRH and DH physiology.

5. Ligand Specificity and Signal Transduction mechanisms

The CRH1 receptor has the highest affinity for CRH as well as Ucn and its orthologues (UI, SVG) and virtually no affinity for Ucns2 and 3 (Figs 2,3). Despite initial postulations that Ucn (and therefore UI) was the cognate ligand for the CRH2 receptor, neuroanatomical studies did not support this supposition (Bittencourt et al., 1999) (Fig 2). In fact, the CRH2 receptor is much more promiscuous than the CRH1 receptor and binds to all vertebrate paralogues, as well as a number of synthetic CRH peptide variants (Hauger et al., 2006; Tellam et al., 2002). The ligand specificity of the receptor reflects its evolution. Ucns 2 and 3 are the product of a direct gene (genomic) duplication, whereas CRH and Ucn (UI, SVG) are the result of a separate direct gene duplication. Thus, their functional distinctiveness, with respect to their receptor affinities is consistent with this evolution (Boorse et al., 2005; Lovejoy and De Lannoy, 2013; Lovejoy and Jahan, 2006). The pharmacological activities among the four CRH-like ligand and their two receptors provide insight into the evolutionary selection pressure on the CRH receptor-ligand co-evolution. Although much data is missing and It is not known when the CRH receptors duplicated, with respect to the vertebrate genome expansion event (2R) are concerned, it is possible to provide a plausible model for CRH ligand and receptor co-evolution. However, given the promiscuous associationa of the R2 receptor with all four CRH –related ligands, we postulate that the CRHR1 and R2 receptors resulted in the first round of genome duplications. In the second round of genome duplications, the new paralogous receptor genes were subsequently lost (Fig 3). Numerous studies have indicated that there may not be only a ligand- and receptor-mediated role to signal transduction but, also that tissues may impart a third level of specificity (Grammatopolous et al, 2000). The C-termini of the CRH family of ligands binds to the extracellular binding pocket of both receptors, whereas their N-termini interacts with the other extracellular loops to activate the intracellular signal cascade (Grace et al., 2004; Hoare et al., 2004). Pharmacological studies of the various CRH homologues established that the C-terminus of the peptide binds to the extracellular 10

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binding region of the CRF receptors whereas its N-terminus stimulates the intracellular signalling cascade via interaction with other regions in the receptor (Perrin et al., 2002). The primary signal transduction system for all CRH receptors characterized to date occurs via the coupling of G-stimulatory (Gs) proteins leading to activation of the adenylate cyclase (AC)-protein kinase A (PKA) pathway (Chen et al., 1993; Hauger et al., 2006; Olianas et al., 1995) (Fig. 2). However, there is considerable evidence that the CRH receptors can also couple with Gq-proteins and other G proteins to stimulate an IP3- and Ca2+mediated signal transduction pathways (Hauger et al., 2006; Guknecht et al., 2010). Moreover, further studies indicate that CRH receptors are coupled to, and activate at least five different G-proteins (Gs, Gi, Gq/11, Go and Gz; Grammatopolous et al., 2001). Some of these effects may be ligand-dependent. For example, in cultured human pregnant myometrial cells, Ucn but not CRH, induce MAPK phosphorylation and activation, suggesting that in the human myometrium, these two peptides have distinct actions and biological roles. In stably-expressed HEK293 and CHO cells, the CRH1a and CRHR2b, but not the CRH1b,c, d, receptor subtypes, mediate Ucn-induced MAPK activation. Activation of Gq, with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation, correlated with MAPK phosphorylation. In these studies, Ucn was 10 times more potent than CRH. Other pathways may also be activated. For example, the ERK1/2 mediated pathway is also activated in a number of in vitro systems (Brar et al., 2004; Hauger et al., 2006).

6. Expression of CRH receptors

Consistent with the early evolution of CRH receptors in the Metazoa are the findings of CRH receptors in diverse tissues that mediate a spectrum of physiological effects. Generally, CRH receptors are associated with the homeostatic actions on cells and organisms with respect to energy metabolism and associated diuretic requirements in response to stressful challenges (Chen et al., 2003; Jannsen and Kozicz, 2013; Lovejoy, 2012; Lovejoy and De Lannoy, 2013). Fundamentally, the CRH peptide and receptor system is associated with sympathetic arousal in vertebrates. Systems associated with parasympathetic activation, (e.g. growth, feeding,digestion), may be expected to be inhibited by CRH activation, whereas sympathetic arousal systems associated with adrenal/interrenal and cardiovascular activity, for example, are more likely to be activated by CRH-associated peptides. Both receptors are expressed in a number of tissues throughout the organism and appear to vary with respect to species and taxa, although information is far from complete. Generally however, the CRH1 receptor is predominantly found in the CNS, whereas the CRH2 receptor, although highly 11

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expressed in the CNS, is found to a greater degree, relative to the CRH1 receptor, in peripheral tissues. If the earliest function of the CRH-DH system is considered and assuming that its initial function was to regulate stressful stimuli associated with diuresis and feeding, then the expression of the CRH receptors would be expected to be found in the tissues and organs of more complex organs associated with these functions. The CRH receptors are expressed early in development in vertebrates. The CRH1 and 2 receptors display distinct patterns of development in fetal brain brains whose expression patterns vary during fetal and postnatal life until becoming more stable in adulthood (Avishai-Eliner et al., 1996; Eghbal-Ahmadi et al., 1998). The hippocampus, hypothalamus and cerebellum have been particularly well studied. For example in rats, CRH2 is expressed in the ventromedial hypothalamus on fetal day 16, prior to the detection of CRH itself in the paraventricular nucleus where it may act to regulate the actions of CRH and paralogues on target neurons (Eghbal-Ahmadi et al., 1998). Moreover, in the developing cerebellum, CRH2a is involved in the survival and differentiation of Purkinje cells and GABAergic neurons, whereas in the adult, CRH2a may act to modulate glia associated with the regulation of cells (Lee et al., 2007). Similar findings in the opossum cerebellum have been reported by Madtes and King (1996). Unfortunately, there are few comparative studies in non-mammals. In the zebrafish, early stage larvae show an expression of both CRH receptors as well as the binding protein, CRH and UI (Alderman and Bernier, 2009). In amphibians, CRH, and its cognate receptors, play a major role in metamorphosis (Denver, 1997) as well as being associated with pituitary thyropin release (Manzon and Denver, 2004). In adult mammals (notably rodents), a number of studies in the brain have indicated that the CRH1 receptor is highly expressed in the cortex, hippocampus, amygdala, olfactory bulb, lateral septum, thalamus, raphe nucleus, pituitary gland and spinal cord (Van Pett et al. 2000) (Fig 4). Among neurotransmitter systems associated with the CNS, the CRH1 receptor is present in glutamatergic neurons of cortex and hippocampus, GABAergic cells in the reticular thalamic nucleus, globus pallidus and septum, dopaminergic neurons of the substantial nigra pars compacta and ventral tegmental area and in 5-HT neurons of the dorsal and medial raphe nuclei (Refojo et al., 2011). The CRH1 receptor is also found in a number of peripheral tissues as well, for example in the female reproductive tract (Nappi and Rivest, 1995; Kiapekou et al., 2011), skin (slomiski et al., 1995), adrenal gland (Squillaciota et al., 2011; Willenberg et al., 2005) and gastrointestinal tract (Chatzaki et al., 2004). Among teleost fishes, the CRH1 receptor is expressed in number tissues. In the Japanese pufferfish, it is found in brain, liver, heart, gonads and to a lesser extent in kidney, gut, and gills (Cardoso

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et al., 2004). In the common carp (Cyprinus carpio) this receptor has been found in integument and gills (Mazon et al., 2006). Overall, the CRH2 receptor shows a more limited expression relative to the CRH1 receptorbased on rodent studies (Lovenberg et al., 1995; Perrin et al., 1995; Van Pett et al., 2000). In comparison to the CRH1 receptor, it is poorly expressed in the cortex and is generally predominately expressed in subcortical regions such as the ventromedial hypothalamus, dorsal raphe nuclei of the midbrain, nucleus of the solitary tract in the hindbrain and various hindbrain nuclei (Bittencourt et al., 2001) (Fig 4). It is also found in a number of regions that also express the CRH1 receptor such as the septal nuclei, although the CRH1 is predominant in the medial septal nuclei whereas the CRH2 is more prevalent in the lateral regions of this nucleus. These findings led, in part, to the original supposition that Ucn (UI, SVG) were the cognate ligand of the CRH2 receptor (Vaughan et al., 1995). However, later studies (Bittencourt et al., 1999), indicated that many of the CRH2-expressing regions of the brain did not receive input from Ucn-containing fibers. In the peripheral regions, the CRH2 receptor has been located to heart (Lovenberg et al., 1995; Perrin et al., 1995; Kishimoto et al., 1995; Stenzel et al., 1995), lung (Lovenberg et al., 1995), gastrointestinal tract (Chatzaki et al., 2004; Perrin et al., 1995), skeletal muscle (Kishimoto et al., 1995), male reproductive system (Perrin et al., 1995) and adrenal gland (Muller et al 2001).

7. Evolution of CRH Receptors

Currently, the literature consistently shows that a single CRH-like receptor (DH) is present in nonchordates (protochordates, arthropods), whereas, two receptors (CRH1, CRH2) are the norm for chordates. In non-chordates, a single CRH-like ligand is associated with its putative cognate CRH-like receptor. There are some lineage specific exceptions. For example, in Ciona, there are two CRHreceptors in the genome, but one ligand (Sherwood et al., 2006; Lovejoy and Barsyste-Lovejoy, 2010), although the two Ciona CRH-like receptors appear to be gene duplications confined to the tunicates (Sherwood et al., 2006). However, in chordates, there are typically four CRH-related ligands, yet only two receptor paralogues. Although the number of ligands is in agreement with the 2R hypothesis, this is not the case with the receptors. A single receptor gene, inherited by the basal chordates, should have diverged into two receptors after the first genomic duplication, and then four after the second genomic duplication. No species have been found with more than two CRH receptors, with the exception of a catfish which, although does possess a third receptor, appears to be the result of a lineage-specific 13

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genome expansion event. As a result, we conducted a detailed in silico analysis of the gene and genomic databases using a BLAST search (Fig 5). Thus, utilizing this methodology, we could only discern a single receptor system present in nonchordates, and two receptors found in chordates consistent with the literature. CRH1 and CRH2 receptors in chordates cluster as distinct clades along the expected phylogenetic lines. Both receptors form a sister lineage to the DH receptors found in invertebrates. However, the presence of only two receptor systems in chordates does not seem to be consistent with the 2R hypothesis (Amores et al., 1998, Mulley et al 2009, Mungpakdee, 2008). Theoretically, four receptor genes should have occurred as a result of the two genomic expansion events (see earlier discussion). One interpretation is that the other set of receptors, resulting from the second gene duplication, were redundant and lost through chordate evolution. This should manifest as the appearance of psuedogenes, yet currently none have been detected. This suggests that the expected paralogues resulting from the second genome expansion event may have been lost early in chordate evolution (Figs 3 and 6). Two CRH-like receptors have been identified in Ciona although they are more similar to each other than either is to the vertebrate CRH1 and CRH2 receptors (Campbell et al., 2004; Sherwood et al., 2006) suggesting that the two genes are likely the result of a gene duplication event in the urochordates and is consistent with the notion of chordates inheriting only a single CRH receptor gene. Thus, given this scenario, we suggest that before the bifurcation of deuterostomes and protostomes, only a single CRH-like ligand, receptor and binding protein were present, which acted as an integrated functional unit to develop into the complex CRH system present in chordates (Fig 6). 8. Functions of the CRH Receptors

Once a new gene or protein becomes selected for a function that increases the fitness of an organism, then it is less likely to change in subsequent generations. The pre-vertebrate evolution of the CRH receptors that occurred before the bifurcation and subsequent development of more complex deuterostome and protostome lineages meant that this receptor-ligand system became well ensconced into numerous physiological circuits spanning diverse tissues (see earlier section). Moreover, its utilization as an endocrine/neuroendocrine system regulating the control of energy metabolism, as a response to homeostatic stressors led to the regulation of tissue and organ physiologies that were ultimately affected by these stressors. In continuation of this trend, a major physiological development in the CRH system in chordates was the formation of a functional HPA system.

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After the CRH peptide became associated with the regulation of the HPA/I axis and the anticipation of the stress-response, it became under a much greater physiological constraint leading to less variation in its structure. This conservation of function is reflected by the pharmacology of the CRH1 receptor which shows greater selectivity for its ligands. Thus, in this respect, the CRH1 receptor is more specialized than the CRH2 paralogue. The formation of the HPA/I axis acted to combine the neural actions of the CRH-peptide with the glucocorticoid-synthesizing tissues of the periphery, thereby linking a comparatively large and mobility-constrained neuropeptide (i.e. CRH) with a small lipophilic hormone (e.g. cortisol/corticosterone) that could pass through all membranes with ease thereby placing the entire organism under the control of a single neuropeptide. In addition, because CRH was under the control of sensory and associative inputs, its regulation allowed the HPA/I to act as a stress-perceiving and integrating unit thereby allowing the anticipation of a future stressor. The earliest functions of the proto-CRH receptors appear to be associated with osmoregulation, which may be considered the most basic and ubiquitous environmental stressors. Indeed, there were several hundred million years of metazoan evolution before animals were capable of surviving in terrestrial environments. Like CRH, UI (Ucn, SV) binds both receptors with physiologically equivalent affinity. In vertebrates, UI became specialized for its role in the urophysis in an analogous manner that CRH became associated with the HPA/I axis. The urophysis or caudal neurosecretory system is analogous to the neurophysis in that it is a neurohaemal organ where the neurosecretory cells release their constituents into the fenestrated capillaries of the vascular system. The caudal neurosecretory system in found in all fishes but has been reduced in the sarcopterygian line and lost entirely in the tetrapods. Thus, this organ system likely evolved in response to adapt to the osmoregulatory stress of rapidlychanging ambient conditions such as salinity that occur in the water column. The urophyseal UIs have been implicated in ion and fluid equilibrium (Lederis et al., 1985) cardiovascular activity (Le Mevel et al., 2006; Platzeck et al., 1998) and can participate in interrenal glucocorticoid release (Arnold-Reed and Balment, 1994; Kelsall and Balment, 1998), all of which are necessary for the adaptability of fishes. Whereas osmoregulatory control may be considered the primary aspect of a stress response, energy regulation in the form of nutrient acquisition, digestion and utilization might be considered the second most important physiology to be protected from a stressor. The role of the CRH ligands and receptors has been studied in detail by numerous authors (Audsley et al., 1997; Spina et al., 1996; Kuperman and Chen, 2008, Zorilla et al., 2003). Recently, studies have suggested that whereas CRH1 may retain much of its osmoregulatory role, the CRH2 receptor, through the actions of its ligands, Ucn 2 and 3, may be more associated with energy regulation including metabolic rate, appetite and feeding 15

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behaviours (Kuperman and Chen, 2008). For example, the ventromedial hypothalamus (VMH) is responsive to changes in circulating glucose and therefore acts, in part, to integrate organismal glucose intake and stores with the needs of the organism. CRH1 activation stimulates the organismal response to low plasma glucose whereas the CRH2 receptor inhibits this response (Cheng et al., 2007; Makino et al., 1999). Mammalian models have been the most studied with respect to CRH receptors and their activity in gastrointestinal (GI) function. Numerous studies in humans, rodents, cats and dogs have established that diverse stressful stimuli induce a variety of actions on the GI tract that typically include delayed gastric emptying and colonic motor activity (Tache et al., 2001; Tache and Bonaz, 2007). Although activation of the autonomic nervous system is responsible for regulation of gastric physiology, CRH receptors in the GI tract play a major role on the acute and chronic actions of stress (Gue et al., 1991; Tache et al., 2001; Williams et al., 1987). The vagus nerve is the main pathway that mediates the delayed gastric emptying and inhibition of gastric motility by CRH and urocortin in rats and dogs. Psychological stress and central administration of CRH and Ucn inhibit small intestine transit and motility via an HPA-independent vagal nerve-associated pathway suggesting a CRH1 receptor mechanism (Kellow et al., 1992; Tache and Bonaz, 2007). Ucn 2, on the other hand, utilizes a CRH2-dependent receptor mechanism that mediates a sympathetic adrenergic receptor system to delay gastric emptying (Czimmer et al., 2006; Martines et al., 2004). The HPA and HPT axes are closely coupled and in some species (fish, amphibians), CRH may act as thyrotropin-releasing factor (Denver, 1997). In chordates, thyroid physiology is required for the maintenance of metabolic rate and regulation against metabolic challenges associated with temperature extremes and general energetic demands. In frogs, CRH2-selective ligands stimulate thyrotropin release from the pituitary, an effect that is blocked by CRH2 specific antagonists, but not CRH1-specific antagonists (De Groef et al., 2006; Okada et al., 2007). In the thyroid gland, CRH1 and 2 receptors are differentially expressed where CRH2 is localized in the C-cells and CRH1 in blood vessels (Squillacioti et al., (2012). Moreover, mice lacking the CRH2 receptor show impaired responses to cold stressors (Bale et al., 2007), a function that is generally attributed to the HPT axis. Assuming that the organism’s osmoregulatory and energetic demands are met, and it can survive various stressful challenges, then arguably the next essential physiology is associated with reproduction. The actions of HPA axis and CRH peptides on the regulation of reproduction has been well documented over numerous studies in the last 30 years (see Chand and Lovejoy, 2010, Lovejoy and Barsyte, 2011; Tellam et al., 2000 for detailed discussions). However, only recently have the roles of the 16

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receptors become apparent. For example, both the CRH1 and 2 receptors modulate aspects of GnRH synthesis, pulsativity and release into the portal blood vessels (Li et al., 2010; Tellam et al., 1998), which may not only affect GnRH and reproduction in the adult, but also regulate the timing of puberty (KinseyJones et a., 2010). However, these actions involve both a direct effect of CRH peptides on GnRH neurons as well as the numerous indirect regulatory systems associated with GnRH release. The CRH1 receptor has been implicated in a number of reproductive processes at the peripheral level. In mouse preantral follicles and oocyte development, CRH inhibits growth and development in vitro likely through a CRH1mediated receptor action (Kiapekou et al., 2011). In monkeys, CRH, Ucn and the CRH1 receptor expression in luteal cells varies as a function of the monkey menstrual cycle suggesting that the CRH system and its requisite receptors act, in part, to regulate follicular growth and development (Xu et al., 2007). In the placenta, the glucose transporters GLUT1 and GLUT3, as well as oestradiol and progesterone biosynthesis are differentially regulated by CRH1 and 2 receptors, where they may be involved in local energy requirements of the placenta during different periods of pregnancy (Gao et al., 2012a,b). The CRH ligand and receptor system has been most intensively studied with respect to both the direct actions of the HPA system and the pathologies associated with its over- or under-activation. At a clinical level, considerable evidence indicates that chronically elevated levels of CRH in the CNS plays a significant role in the aetiology of a number of affective and neurological disorders, thus there has been much interest in understanding the regulatory mechanisms of the CRH receptors. This has been reviewed in detail by a number of authors (Dunn and Berridge, 1990; Hauger et al., 2006; Janssen and Kozicz, 2013; Mitchell, 1998; Rotzinger et al., 2010). In clinical and mammalian model systems, both receptor systems are implicated in different elements of stress-associated pathologies. However, establishing a clear action for either receptor is confounded by the difficulty in using behavioural models and situations that mimic the human situation. Numerous studies using selective CRH receptor agonists and antagonists indicated that generally, a stressor needs to be present in these models to observe a clear effect (see Rotzinger et al., 2010 for a detailed discussion). Recently, the utilization of receptor knockout studies has revealed considerable insight into the specific actions of CRH receptors with respect to stress-associated conditions and pathologies. CRH1-specific knockout mice show a reduced response to stressors with slightly elevated ACTH and glucocorticoid levels (Smith et al., 1998), whereas CRH2-deficient mice possess a generally normal initiation of the stress-response, but show an early termination of the HPA-associated ACTH release and possess an impaired cardiovascular response,

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although do not show changes in the stress-response using some behavioural models (Coste et al., 2000; 2006). Despite the difficulties relating the human and mammalian experience to non-mammalian models, there are a number of studies that suggest, at least with respect to ‘emotional’ reactivity, the CRH receptor system has a similar function. For example, among fishes, rainbow trout (O. mykiss) are particularly sensitive to environmental stressors. The high (HR) and low responder (LR) rainbow trout strains have been particularly useful to understand the role of CRF and HPA/I physiology in fishes and non-mammals in general. After confinement, HR trout showed significantly decreased CRH2 in forebrain mRNA that the LR trout, although there was no difference in CRH1 transcript levels (Backstrom et al., 2011). In the crucian carp (Carassius carassius) the CRH1 antagonist, antalarmin, reduced the olfactorymediated fright reaction leading to a decrease in cortisol levels (Lastein et al., 2008).

9. Stress Response and Complementarity of the CRH receptors in Chordates Our understanding of the interaction of the CRH1 and 2 receptors have undergone a number of interpretations. Risbrough and Stein (2006) have summarized two prevailing theories on the role of the CRH receptors. One such concept posits that the CRH2 receptors facilitate the recovery of the stressresponse by inhibiting the initial CRH1-mediated stress response. A second hypothesis suggests that under chronic stress, the CRH2 receptors could act to facilitate “depressive-like” behaviour over defensive behaviours typically mediated by CRH1 activation (see Coste et al., 2006; Hammack et al., 2003; Koob and Heinrichs, 2004). In essence, both of these hypotheses are similar in that both suggest that there are two distinct physiological aspects to the stress response. The initial phase, perhaps coinciding with Selye’s (1950) alarm reaction, which as he termed it, is a ‘call-to-arms’ to mediate the homeostasis challenging actions of the stressor, is mediated by the CRH1 receptor. A second phase, which Selye (1950) termed ‘stage of resistance’ is the result of secondary physiological mechanisms activated in response to prolonged or chronic stress. Indeed, we have previously argued that because ‘depressive’ like behaviours are found in a number of species, that this is an evolutionarily conserved response to remove the organism from continued stressful stimuli by sensory withdrawal (Lovejoy and Barsyte, 2010). In humans, and defined by Selye’s (1950) ‘stage of exhaustion’, may be exemplified as clinical depression and suicide in extreme situations. In non-humans, this behaviour would be manifested by social withdrawal and hiding. Thus, these theories of the actions of the CRH receptors are consistent with our classical understanding of the regulation of the stress response.

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However, recently Jannsen and Kozicz (2013) have challenged this view of the respective actions of the receptors, whilst respecting Selye’s (1950) original hypothesis. They have argued that stressful stimuli impinges upon regions of the brain to activate particular neurotransmitter-specific regions (glutamate, GABA, dopamine, 5-HT) depending upon the specific challenge leading to an appropriate stress-associated response that may be mitigated by either CRH1 or 2 receptors. For example, citing the study by Refojo and associates (2011), CRH1 knockouts in various transmitter systems in specific regions of the brain, may lead to anxiolytic- or anxiogenic-like behaviours depending upon the behavioural model used for analysis (see above). From an evolutionary point of view, this theory has merit. Two rounds of genomic duplication in the chordates have a complex effect on the neurological organization, and indeed, the physiology of the organism. Such widespread genetic expansion events such as expected in an entire genomic duplication is likely to have a profound affect not only on the requisite genes, but also on the subsequent development of the entire organism. In the brain, this may manifest as novel nuclei, new inter-neuronal connections, and interaction with peripheral organs. Moreover, it is not understood what the relationship is between neuronanatomical development and the formation of novel paralogue formation of potent neuromodulators such as CRH. Neurotransmitter systems such as glutamate, GABA, dopamine and 5-HT evolved well before peptide modulators such as CRH (see Lovejoy, 2005 for discussion) indicating that neuronal networks were well established in the Metazoa before they had the capacity to be modulated by more complex sensory experiences. Conclusions The evolution of the CRH receptors as a functional system integral to diuresis and nutrient regulation in the early Metazoa subsequently led to their widespread expression among the tissues and organs of diverse metazoan species. In chordates, inheritance of this proto-CRH ligand-receptor system was the subject of two rounds of genome duplications which led to expansion of function. One of these paralogues, the CRH1 receptor became associated with the hypothalamic-pituitary-adrenal/interrenal axis to coordinate the organismal stress response and became specialized for its interaction with CRH and urotensin-I (UI) and its mammalian orthologue urocortin (Ucn). The CRH2 receptor remained less specialized to bind and continued to activate all four ligand paralogues (CRH, UI/Ucn, Ucn2 and Ucn3. Both receptors work in tandem to regulate elements of the organismal stress response in vertebrates, but may maintain tissue-specific actions. Coordination of both receptor systems may be regulated by higher neurological systems, for example the autonomic nervous system.

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Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding: This work was supported by the Canadian Natural Sciences and Engineering Research Council (grant number 458080, 2010).

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Figure Legends

Figure 1: Human CRH paralogues. CRH and urocortin consist of one paralogous lineage, whereas urocortin 2 and 3 comprise the second paralogous lineage in chordates. Figure 2: Scheme of the molecular interaction among CRH ligands, receptors and the binding protein in chordates and insects. See text for discussion. Figure 3: A model for the CRH peptide and receptor co-evolution in chordates. A single ligand and receptor gene present in the protochordate genome was inherited by the early chordates. The first round of genome duplication led to the initial expansion of ligand and receptor into two paralogues. Over time, the receptors evolved into early R1 and R2 forms, whereas the two peptide genes evolved into either CRH/UI-like or Ucn 2/3 like forms. The next genome duplication created the divergence of CRH-like peptides into CRH and UI/ Ucn /SVG and Ucn 2 and Ucn3 forms whereas the receptors diverged into R1 and R2 forms. We postulate that the redundant R1 and R2 paralogues were subsequently lost and, therefore, only 2 CRH receptors were retained by modern chordates. Legend: CRH: corticotrophin releasing hormone, UI: urotenins I, Ucn: urocortion, Ucn2: urocortin 2, Ucn3: urocortin 3. The black lines and arrows indicate the evolutionary direction and selection. Coloured arrows matching the ligand indicate the affinity for the receptors. Figure 4: Major regions of CRH1 and CRH2 expression in rat brain. Key: AM; amygdala, AN; arcuate nucleus, AO; accessory olfactory bulb, AP; anterior pituitary gland, BN; bed nucleus, CB; cerebellum, CP; caudate putamen, CT; cortex, DR; dorsal raphe nucleus, HF; hippocampal formation, IC; inferior colliculus, IP; intermediate lobe of the pituitary gland, OB; olfactory bulb, PB; parabrachial nucleus, PN; paraventricular nucleus, PO; pontine nuclei, SC; superior colliculus, SN; septal nucleus, ST; nucleus of the solitary tract, SU; substantia nigra, VH; ventromedial hypothalamus, VT; ventral tegmental area.

Figure 5. Maximum likelihood phylogeny of CRF-like proteins. CRH-like protein sequences were obtained via BLAST searches of the NCBI sequence databased, aligned using ClustalW (Thompson et al. 1994, Larkin et al. 2007), and trimmed by hand to eliminate regions of uncertain alignment. The trimmed data set was then subjected to phylogenetic analyses using maximum likelihood and Bayesian 33

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methods (Guindon and Gascuel 2003, Ronquist and Huelsenbeck 2003, Guindon et al. 2010). Maximum likelihood phylogenetic methods were implemented in the program PHYML 3.0 (Guindon and Gascuel 2003, Guindon, et al. 2005), using the LG amino acid replacement matrix (Le and Gascuel 2008). For the likelihood analyses, node support was assessed using an approximate likelihood-ratio test (aLRT, (Anisimova and Gascuel 2006)). Bayesian inference was performed in MrBayes 3.1.2, using a model that allows for jumping among fixed amino acid substitution rate matrices (Ronquist and Huelsenbeck 2003), with all of the protein sequence data in a single partition. Two Markov chain Monte Carlo runs were performed, with four chains each (three heated and one cold) for 1 million generations. Convergence was assessed using standard methods, including the average standard deviation of split frequencies, and the potential scale reduction factor (PSRF, (Gelman and Rubin 1992)). The first 25% of trees sampled were discarded as burn-in, and remaining trees were taken as representative of the posterior probability distribution (Fig 4).Node is support indicated by likelihood aLRT values, and Bayesian posterior probabilities (italics). CRH1 and CRH2 receptors cluster as discrete groups and represent, together a sister lineage of the insect DH receptors. Legend: (species abbreviation, species name and accession name

is

indicated):

ADOME:

Achedta

domesticus,

Q16983.1;

AAEGYI:

Aedes

aegypti,

ABX57919.1;Ameiu: Ameirurus nebulosus AAK01069;Anoli: Anolis carolensis, XP0032211923; APISU A: Acyrthosiphon pisom, XP003244979.1 APISU B: XP001944842.2;

BMALA: Brugia malayi, XP

001899608.1; BMORI: bombyx mori, XP004933474.1; Bos R1, R2: Bos Taurus, NP776712, NP001179474; BTERR: Bombus terrestris XP003394723.1; Calli: Callithrix jactaus, XP002748148; CGIGA: Crassotrea gigas, EKC3340.1; CINTE: Ciona intestinalis, XP002123381.1; CQUIN: Culex quinquefaciatus, DAA06284.1;CSINE: Clonorchis sinensis, GAA51272.1; Danio: Dano rerio, XP696346; DMELA1, A2, Drosophila melanogaster NP610960.1, NP725175.3; DVIRIA, B: Drosophila virilise XP002059297.1, XP002050193.1;

Gallu RI, R2: Gallus gallus NP989652; NP989785; Haplo: Haplochromis burtonii,

ACV53954; ISCASP_ PU, _HP: Ixodes scapularis XP002403968.1, XP002403764.1; HomoR1, R2: Homo sapiens NP001138618;

ABV59317;

Macac: Macaca mulatta,

EHH17404,

MOCCI: Metasluslus

occidentalis, occidentalis, XP002123381.1; Monod R1, R2: Monodelphis domestica, XP001375959, XM001373511.2; Mus: Mus musculus Q60748; Muste: Mustela putorius furo, XP004762632; NLUGE: Nilaparvata lugens, CA625575.2; Octod: Octodon degus, XP00466578; Oncor: Oncorhynchus keta, CAC81754: Orcin R1, R2: Orcinus orca, XP004275734, XP004269989;

PHUMA; Pediculus humas

corporis, XP002424517.1; Ptero: Pteropus alecto, ELK12633; Rana: Rana catesbiana, BAD36784; Rattu: Rattus norvegicus, NP112261; Salmi: Saimirii boliviensis boliviensis, xp003942463;

SKOWA:

Saccoglossus kowalevskii, NP 001161520.1; SPURP: Stongylocentrotus purpurati, XP790450.3; TCAST: 34

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Tribolium casteneum, NP001167548.1; TSPIR: Trichinella spiralis, XP003376880.1; Tursi R1, R2: Tursiops truncatus, XP004318934, XP004314441. Figure 6: Evolution and structure function relationships of CRH receptors. At the origin, only a single ligand, receptor and binding protein was present. Two rounds of gene expansion events in chordates led to the formation of four ligands, two receptors, but only a single binding protein.

35

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Corticotropin Releasing Hormone Urocortin

(NM000756) (NM003353)

SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-NH2 .DNPSLSIDLTFHLLRTLLELARTQSQRERAEQNRIIFDSV-NH2

Urocortin 2 Urocortin 3

(BC022096) (NM053049)

...IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV-NH2 ...FTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2

Page 37 of 41

Vertebrate

Insect BP

BP

CRH

Gqα

UI Ucn Svg

Ucn 2

Ucn 3

Gα CRH1

DH

Gqα CRH2

Gqα

? DHR

Gα

Page 38 of 41

CRH ligand

CRH receptor

Protochordates 1st genome duplication

CRH/UI like

Ucn 2,3 like

R1- like

R2- like

Early chordates 2nd genome duplication CRH-like

CRH

UI-like

Ucn2like

Ucn3like

R1

R2

UI, Ucn SVG

Ucn2

Ucn3

R1

R2

Modern chordates

Page 39 of 41

CT HF OB

SC

SN

CP

IC

PT PN AO CRH1 CRH2

VH

BN/AM OT

CB DR SU/VT PO

AN AP/IP

PB

ST

Page 40 of 41

CSINE 0.94

1

0.99

SPURP TSPIR

1

BMALA_PR CGIGA

APISU_A NLUGE1PU TCAST_BPU PHUMA_PU BTERR_A

0.31 –

0.91 0.77

APISU_B

0.87

ADOME_PR

–

MOCCI ISCAP_PU ISCAP_HP

1 0.99

0.3

Arthropod

TCAST_A BMORI DMELA2A DVIRI_A DMELA1 DVIRI_B CQUIN

– 0.98 1

AAEGYI SKOWA CTELE_HP CINTE_C 0.71 0.77 0.54 0.61

0.95 1

0.88 1

0.99 1

0.93 1 0.81 0.92

Rana_CR1 Gallu_CR1 Monod_CR1 Rattu_CR1 1Homo_CR1 Saimi_CR1 Bos_CR1 Tursi_CR1 Orcin_CR1 Anoli_CR1 Danio_CR1 Oncor_CR1 Haplo_CR1 Rana_CR2 Anoli_CR2 Gallu_CR2 Ameiu_CR2 Haplo_CR2 Oncor_CR2 Monod_CR2 Muste_CR2 Orcin_CR2 Tursi_CR2 Ptero_CR2 Bos_CR2 Mus_CR2 Oryct_CR2 Octod_CR2 Calli_CR2 Homo_CR2 Macac_CR2

Vertebrate CR1

Vertebrate CR2

2.0

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Chordates

Tunicates Arthropods

Other groups

? Deuterostsomes

CRH ligands Receptors Binding protein

Protostsomes